Caspases,Paracaspases, and Metacaspases Methods and Protocols

168 Wash the stained cells with PBS twice. Add 0.3 mL PBS, protect samples from light, and measure the FITC fl uorescence (FL1- ...

169 4 Notes To avoid SDS precipitation, the sample buffer needs to be heated prior to use. Initially, concentrated HCl (12 N) c ...

170 Protein fragments produced as a result of caspase-mediated cleavage of specifi c substrates are not always possible to dete ...

171 (c) Remove stain by repeated washing in water or PBS. (d) Proceed to the blocking procedure. To minimize evaporation and th ...

172 Prepared samples can be stored in methanol at −20 °C for several weeks. To prevent clustering of cells which could interfer ...

173 apoptosis induction in a particular cell type. Moreover, once initiated, apoptosis is normally a fast process which in cell ...

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Part II Paracaspases and Metacaspases ...

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177 Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols, Methods in Molecu ...

178 with caspases and metacaspases [ 2 ]. However, MALT1 showed no caspase-like activity [ 2 , 12 ]. Therefore, it was initially ...

179 cleavage of A20 might serve to prolong the IKK-dependent NF-kB response [ 17 ]. However, pretreatment of cells with the MALT ...

180 of tetrapeptides in which the three amino acids (P2–P4) preceding the fi xed C-terminal Arg residue were systematically vari ...

181 2 Materials Deionized water is used throughout. All reagents are prepared and stored at room temperature (unless indicated o ...

182 TEMED. Resolving buffer: 1.5 M Tris–HCl pH 8.8. Reporter plasmid: constructed by linking eYFP and eCFP with the MALT1 cleav ...

183 Pellet the bacteria by centrifugation at 3,700 × g for 20 min at 4 °C. Resuspend the pelleted bacteria with 15 ml of lysis ...

184 Measure the fl uorescence (excitation 350–380 nm, emission 460 nm) over time (measure every 10 min for 2–4 h) at 30 °C. To ...

185 Incubate the mix for 10 min at room temperature. Distribute the transfection mix carefully and equally on the cells. Change ...

186 when GST-MALT1 is bound to glutathione-coupled beads. This approach might be considered if the planned experiment is not com ...

187 and B cell antigen receptor signaling. Immunity 19(5):749–758 Ruefl i-Brasse AA, French DM, Dixit VM (2003) Regulation of N ...